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Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus
ARTICLE IN PRESS Neurochemistry International ■■ (2015) ■■–■■
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Rapid communication
Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus Q1 André Serpa a, Isa Pinto a, Liliana Bernardino a, José F. Cascalheira a,b,* a b
CICS-UBI – Health Sciences Research Center, University of Beira Interior, Covilhã, Portugal Department of Chemistry, University of Beira Interior, Covilhã, Portugal
A R T I C L E
I N F O
Article history: Received 19 April 2015 Received in revised form 3 June 2015 Accepted 4 June 2015 Available online Keywords: Adenosine A1 receptor Cannabinoid CB1 receptor Excitotoxicity Hippocampus
A B S T R A C T
Both adenosine A1 and cannabinoid CB1 receptors trigger similar transduction pathways and protect against neurotoxic insults at the hippocampus, but their combined neuroprotective potential has not been investigated. We set forth to assess the combined action of A1 and CB1 receptors against glutamate NMDA receptor-mediated excitotoxicity at the hippocampus. Cell damage was assessed by measuring propidium iodide (PI) uptake and lactate dehydrogenase (LDH) activity released from cultured hippocampal slices exposed to NMDA. Exposure to NMDA (50 μM) for 1 h increased LDH activity by 92% ± 16%. WIN55212-2 (30 μM), a cannabinoid CB1 receptor agonist, decreased NMDA-induced LDH activity by 53% ± 10% while N6-cyclopentyladenosine (CPA, 100 nM), an adenosine A1 receptor selective agonist, decreased it by 37% ± 11%. The combined inhibitory effect of WIN55212-2 and CPA (88% ± 12%) did not differ from the sum of the individual inhibitory effects of each agonist (90% ± 15%) but was different from the effects of CPA or WIN55212-2 alone. An additive inhibitory effect of co-application of WIN55212-2 (30 μM) and CPA (100 nM) on NMDA (50 μM)-induced PI uptake was also observed in CA3 but not in CA1 area of the hippocampal slice. The results suggest that both CB1 and A1 receptors produce additive cumulative neuroprotection against NMDA-induced excitotoxicity in the hippocampus. © 2015 Published by Elsevier Ltd.
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1. Introduction Sustained activation of glutamate NMDA receptors, following traumatic or ischemic brain injury, or caused by the pathological mechanisms of neurodegenerative diseases, generates neuronal death due to excitotoxicity, a secondary brain damage mechanism which involves excessive release of the neurotransmitter glutamate (Cross et al., 2010). The Gi/o-protein coupled cannabinoid CB1 and adenosine A1 receptors are both expressed at high levels in the hippocampus, were they inhibit glutamatergic synaptic transmission (Serpa Q2 et al., 2009) and protect against neurotoxic insults (Sebastião et al., 2001; Zhuang et al., 2005). Given the similarity between transducing pathways operated by adenosine A 1 and cannabinoid CB 1 receptors, as well as the identical effects produced by both receptors on nerve cells, clarification of the combined activity of these receptors is particularly relevant, since the harmful effects of neurotoxic insults might be cumulatively dampened. Previous studies showed that acute co-activation of adenosine A1 and cannabinoid
* Corresponding author. Departamento de Química, Universidade da Beira Interior, Rua Marquês D’Ávila e Bolama, 6200 Covilhã, Portugal. Tel.: +351 275329259; fax: +351 275319730. E-mail address: [email protected] (J.F. Cascalheira).
CB1 receptors generates additive effects when inhibiting excitatory synaptic transmission and cAMP formation in the rat hippocampus (Serpa et al., 2009, 2015). We now further tested how the combined action of A1 and CB1 receptor agonists modulates NMDA-mediated excitotoxic insult at the rat organotypic hippocampal slice.
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2. Materials and methods 2.1. Organotypic hippocampal slice cultures The interface culture method was used to prepare hippocampal slice cultures from 7-day-old Wistar rats (for details see Bernardino et al., 2005). Animals were handled according to European Community guidelines and Portuguese law concerning animal care. Slices (350 μm coronal sections) were prepared and sets of six were positioned on porous (0.4 μm) insert PTFE membranes (30 mm diameter; Millipore Corp., Bedford, USA) and transferred to a 6-well culture tray (Corning Costar, Corning, NY, USA). To each well was added 1 mL culture medium, composed of Opti-minimal essential medium (50%), heat-inactivated horse serum (25%), Hank’s balanced salt solution (25%) (all from Invitrogen, Paisley, UK), supplemented with D-glucose (25 mM) and also contained penicillin
http://dx.doi.org/10.1016/j.neuint.2015.06.005 0197-0186/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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(100 U/mL) and streptomycin (100 μg/mL) (Invitrogen). Culture trays were incubated at 37 °C under 5% CO2 and 95% atmospheric air and culture medium was changed twice a week for 2 weeks. Twentyfour hours before the experiment, medium was replaced with 1 mL serum free Neurobasal medium containing L-glutamine (1 mM) and 20 μL B27 supplement (Invitrogen). Previous work showed that 30 μM WIN55212-2 and 100 nM CPA produced a nearly maximal activation of CB1 and A1 receptors, respectively, as assessed by inhibition of cAMP formation in the rat hippocampal slice (Serpa et al., 2015), and therefore we used these concentrations to test the agonist neuroprotective effects.
and the PI uptake that would be expected in the presence of CPA, WIN55212-2 and NMDA if the effect of CPA and WIN55212-2 were additive was calculated as: A + B − C; where A, B and C are, respectively, LDH activity/PI uptake obtained in the presence of CPA + NMDA, in the presence of WIN55212-2 + NMDA, and in the absence of CPA and WIN55212-2 but in the presence of NMDA. Significance of differences between mean values obtained in two different conditions, or when comparing means with zero, was evaluated by Student’s t-test. When more than two different conditions were simultaneously compared one-way ANOVA was used, followed by LSD post-hoc test.
2.2. Evaluation of NMDA-induced excitotoxicity in slice cultures
3. Results
At the end of each experiment, aliquots of medium were collected to assess LDH activity. LDH activity was measured at room temperature using a commercial kit (LDH cytotoxicity assay kit, Cayman Chemical, Ann Arbor, USA). Quantification of LDH activity was not affected by endogenously released NADH/NADPH, since failure to add lactate to the reaction solution abolished LDH activity. Cell damage in slice cultures was also evaluated by measuring the cell uptake of propidium iodide (PI). PI (2 μM final concentration) was added to the incubation medium 3 hours before exposure to drugs, for determination of basal PI uptake, and PI addition was renewed after subsequent medium changes. Cellular uptake of PI was assessed by measuring emitted fluorescence (630 nm; absorbance 493 nm) using an inverted fluorescence microscope (Axio Observer Z1, Carl Zeiss, Jena, Germany) equipped with a rhodamine filter and digital camera (F-View 2; Olympus, Hamburg, Germany) with 100 ms exposure time. Fluorescence microscopy images of each slice were digitally recorded before drug exposure (basal PI uptake) and 24 h after exposure to the NMDA challenge (total PI uptake). To quantify cell damage, the slice areas of interest were delineated (>90% of the entire area) using Image J program (1.47 g, NIH, USA) and densitometric measurements of the PI uptake were performed. To assess the maximal level of cell death slices were exposed to an adverse environment (4 °C) for 48 h after performing the experiments. Maximal level of cell death was determined with both PI uptake, for each hippocampal area, and LDH release.
3.1. Neuroprotective action of adenosine A1 and cannabinoids CB1 receptors co-activation on NMDA-induced cytotoxicity in the whole hippocampal slice assessed by LDH release Exposure to NMDA alone (50 μM) for 1 h increased the released LDH activity from 7.0 ± 1.3 nmol/min/mL (control) to 12.4 ± 2.3 nmol/min/mL (19% ± 4% of maximal LDH release) corresponding to a 92% ± 16% increase (n = 4, see Fig. 1). Further application of the cannabinoid CB1 receptor selective agonist WIN55212-2 (30 μM), from 45 min before the NMDA insult, decreased the NMDAinduced release of LDH activity by 53% ± 10% (n = 4). In the same way, the adenosine A1 receptor selective agonist CPA (100 nM), when
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2.3. Drugs The drugs (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55212-2), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), N6-cyclopentyladenosine (CPA), N-(piperidin-1-yl)-5-(4-iodophenyl)1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and N-methyl-D-aspartate (NMDA) were purchased from Tocris (Bristol, UK). Propidium iodide was purchased from Sigma. Stock solutions of WIN55212-2 (20 mM), DPCPX (50 μM) and AM251 (5 mM) were prepared in dimethyl sulfoxide (DMSO) while CPA (2 mM) and NMDA (10 mM) stock solutions were prepared in water. 2.4. Data analysis The values are expressed as mean ± SEM from n experiments. The effect of drug(s) on the NMDA-induced LDH activity was calculated, for each experiment, as: 100 × (N − D)/(N − C); where N is the LDH activity obtained in the presence NMDA, D the LDH activity obtained in the presence of the drug(s) plus NMDA and C is the LDH activity obtained in the control assay performed in the same conditions but in the absence of the drug(s) and NMDA. PI uptake obtained in the presence of drug(s) was expressed as percentage of the PI uptake corresponding to control slices incubated in the same conditions but in the absence of drug(s). The released LDH activity
Fig. 1. Combined neuroprotective effect of WIN55212-2 and CPA on cell death induced by NMDA insult at the rat hippocampus evaluated by released lactate dehydrogenase (LDH). Two weeks old cultured organotypic hippocampal slices were exposed to WIN55212-2 (30 μM) or its vehicle (control). After 30 min CPA (100 nM final concentration) or its vehicle (control) were applied and 15 min later NMDA (50 μM final concentration) or its vehicle (control) were added and incubation continued for 1 h. Medium was replaced to remove drugs and released LDH activity was quantified 24 h later. In each experiment five parallel assays were performed, corresponding respectively to: incubation with NMDA, NMDA + WIN55212-2, NMDA + CPA, NMDA + WIN55212-2 + CPA and incubation in the absence of drugs (control). Bars represent released LDH activity obtained (from left to right) in the absence of drugs (control), in the presence of NMDA (b), NMDA + WIN55212-2 (c), NMDA + CPA (d) and NMDA + WIN55212-2 + CPA; the dashed bar represents the released LDH activity that would be obtained if the effects of CPA and WIN55212-2 were additive. Data are mean ± SEM from 4 independent experiment runs at least in triplicate. *, **, ***: P < 0.05, P < 0.02 and P < 0.00005, respectively, vs NMDA (b) alone (oneway ANOVA followed by LSD test). #, ##: P < 0.05 and P < 0.02, respectively, vs WIN55212-2 (c) or CPA (d) alone (one-way ANOVA followed by LSD test). NS, non statistically different (P > 0.90, Student’s t-test).
Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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applied 15 min prior to NMDA, decreased the NMDA-induced release of LDH activity by 37% ± 11% (n = 4). Interestingly, when WIN55212-2 (30 μM) and CPA (100 nM) were applied together, prior to NMDA (50 μM), the released LDH activity was reduced nearly to the levels obtained in the absence of NMDA (see Fig. 1). The combined application of WIN55212-2 (30 μM) and CPA (100 nM) produced an 88% ± 12% (n = 4) inhibition of the NMDA-induced LDH release from cultured hippocampal slices, which was higher than the inhibition produced by either WIN55212-2 (30 μM) or CPA (100 nM) alone (see Fig. 1) but not different from the sum of the individual inhibitory effect of each agonist (90% ± 15%; n = 4), indicating additivity of effects of both agonists. Neither WIN55212-2 (30 μM) nor CPA (100 nM) modified basal (control) release of LDH activity. The presence of the adenosine A1 receptor selective antagonist DPCPX (50 nM) reduced the inhibitory effect of 100 nM CPA to 1% ± 5% (n = 3, P < 0.05, Student’s t-test, compared with the effect in the absence of DPCPX), whereas the cannabinoid CB1 receptor selective antagonist AM251 (10 μM) decreased the effect of 30 μM WIN55212-2 to 11% ± 2% (n = 3, P < 0.05, Student’s t-test, compared with the effect in the absence of AM251), indicating that the inhibitory effects of CPA and WIN55212-2 are mediated by adenosine A1 and cannabinoid CB1 receptors, respectively. 3.2. Combined neuroprotective action of adenosine A1 and cannabinoids CB1 receptors on NMDA-induced cell injury in different areas of the hippocampal slice evaluated by fluorescence microscopy Fig. 2A depicts the effect of NMDA (50 μM) application for 1 h on the PI uptake in different areas of the rat hippocampal slice; the areas analyzed were those defined by Amaral and Witter (1989). As can be observed (Fig. 2A), NMDA-induced cell injury was highest in the CA3 area, where the PI uptake obtained was increased to 291% ± 19% (32% ± 3% of maximal PI uptake, n = 4) of the control PI uptake obtained in the absence of NMDA, while for the CA1 area the PI uptake was increased to 187% ± 22% of the control (20% ± 2% of maximal PI uptake, n = 4). No significant effect of NMDA (P > 0.05) on PI uptake was observed in CA4 and dentate gyrus areas (Fig. 2A). As can be observed in Fig 2B, for the CA3 area, WIN55212-2 (30 μM) and CPA (100 nM) when applied simultaneously produced a decrease of the NMDA (50 μM)-induced PI uptake, which was higher than the decrease produced by either WIN55212-2 (30 μM) or CPA (100 nM) alone but which was not different from the sum of the individual effects of each agonist. Note that this effect of WIN55212-2 and CPA was specific for the CA3 area. In CA1 area, the PI uptake obtained in the presence of NMDA (50 μM) alone (187% ± 22% of control PI uptake obtained in the absence of NMDA, n = 4) was not different from the PI uptake obtained in slices incubated with NMDA but in the presence of WIN55212-2 (30 μM), CPA (100 nM) or WIN55212-2 (30 μM) plus CPA (100 nM), respectively 182% ± 22%, 214% ± 20% and 180% ± 19% of the control PI uptake (P > 0.05, when compared with NMDA alone, one-Way ANOVA followed by LSD test). 4. Discussion The present study provides evidence, for the first time, of an independent, cumulative inhibitory effect of adenosine A 1 and cannabinoid CB1 receptors against NMDA-mediated excitotoxic insult at the rat hippocampus. Previous studies showed that, when applied alone, both A1 and CB1 receptor agonists decrease glutamatedependent neuronal activity and injury in the hippocampus (Sebastião et al., 2001; Zhuang et al., 2005). In the present work we observed that both A1 and CB1 agonists not only had individual neuroprotective action but also cumulatively dampened NMDAmediated excitotoxicity, with an additive combined effect higher than the one obtained activating each receptor alone. These results are
Fig. 2. Combined neuroprotective effect of WIN55212-2 and CPA on cell injury produced by NMDA insult at the rat hippocampus assessed by propidium iodide (PI) uptake. Two weeks old cultured organotypic hippocampal slices were exposed to WIN55212-2 (30 μM) or its vehicle (control). After 30 min CPA (100 nM final concentration) or its vehicle (control) were applied and 15 min later NMDA (50 μM final concentration) or its vehicle (control) were added and incubation continued for 1 h. Medium was replaced to remove drugs and incubation continued for a 24 h period. PI (2 μM final concentration) was added 3 hours before exposure to drugs and after medium replacement. In each experiment five parallel assays were performed, corresponding respectively to incubation with NMDA, NMDA + WIN55212-2, NMDA + CPA, NMDA + WIN55212-2 + CPA and incubation in the absence of drugs (control). Cellular uptake of PI was assessed by measuring emitted fluorescence before drugs addition (basal PI uptake) and 24 h after exposure to NMDA (total PI uptake). After subtracting basal PI uptake to total PI uptake, the net PI uptake was expressed as % of the net PI uptake obtained for control slices. (A) Bars represent PI uptake obtained in the absence (open bars) or in the presence of NMDA (solid bars) corresponding to (from left to right) the CA3, CA1, CA4 and dentate gyrus (DG) areas of the rat hippocampal slice. (B) Bars represent PI uptake obtained (from left to right) in the absence of drugs (control), in the presence of NMDA, NMDA + WIN55212-2, NMDA + CPA and NMDA + WIN55212-2 + CPA; the open bar represents the PI uptake that would be obtained if the effects of CPA and WIN55212-2 were additive. Data are mean ± SEM from 4 independent experiment runs at least in triplicate. §, §§, §§§: P < 0.05, P < 0.01 and P < 0.0001, respectively, vs NMDA (b) alone (oneway ANOVA followed by LSD test). #: P < 0.05 vs NMDA + WIN55212-2 (c) or NMDA + CPA (d) (one-way ANOVA followed by LSD test). NS, non statistically different (P > 0.9, Student’s t-test). *, **; P < 0.05 and P < 0.0001, respectively, vs control (Student’s t-test).
Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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1 in agreement with previous studies where acute co-activation of 2 adenosine A1 and cannabinoid CB1 receptor have been shown to generate additive effects in inhibiting glutamatergic synaptic trans3 mission and cAMP formation in the rat hippocampus (Serpa et al., 4 2009, 2015). 5 The combined additive neuroprotective action of adenosine A1 6 and cannabinoid CB1 receptors on NMDA-induced excitotoxicity was 7 evident when cell injury was assessed either by measuring LDH 8 release – which reflects cell necrosis and late apoptosis – or quan9 tifying PI uptake – which measures mainly cell necrosis and late 10 not only apoptosis but also early apoptosis in a smaller degree 11 (Parhamifar et al., 2013; Vitale et al., 1993). Although the com12 bined application of WIN55212-2 and CPA nearly prevented NMDA13 induced LDH release from hippocampal slices (88% decrease of LDH 14 release), the decrease in NMDA-induced PI uptake in CA3 area pro15 duced by both agonists was lower (47%). This difference might result 16 from the fact that LDH release reflects cell injury occurring in the 17 whole hippocampal slice, while PI uptake analysis is restricted to 18 superficial cell layers – less exposed to protective/trophic factors 19 released by neighboring cells than cells in the bulk of the slice – 20 that might have a lower ability to recover from NMDA-induced 21 damage upon activation of A1 and CB1 receptors. 22 Using PI fluorescence microscopy we were able to analyze NMDA23 induced cell injury in different hippocampal regions. The NMDA24 induced excitotoxic effect varied across different hippocampal areas, 25 the NMDA effect being stronger in CA3 area than in the CA1 area, 26 while no excitotoxic effect was observed at either CA4 or dentate 27 gyrus, indicating different susceptibilities of the neuronal popula28 tions across different hippocampal areas. The extent of NMDA29 induced cell injury in CA3 and CA1 areas observed in the present 30 work was similar to that reported in Sprague–Dawley rats in a pre31 vious study using the same NMDA concentration and exposure 32 period (Mayer et al., 2002), however they reported a significant neu33 rotoxic effect of NMDA in dentate gyrus, which was not observed 34 neither in the present work nor in a previous study also using Wistar 35 rats (Adamchik and Baskys, 2000). The higher extent of NMDA36 induced cell damage in CA3 than in CA1 area observed in the present 37 work and by Mayer et al. (2002), in contrasts with most previous 38 studies reporting CA1 area as the most susceptible to NMDA39 40 Q3 induced cytotoxicity – see Bruce et al. (1995), Ikegaya and Matsuki Q441Q5 Q6 (2002), Kristensen et al. (2001) and Vornov et al. (1991) (reviewed and cited respectively as references [9], [232], [11] and [12] 42 in Noraberg et al., 2005). The discrepancies between the results ob43 tained by the different studies may probably reflect differences in 44 the NMDA insult protocol, rat strains and/or composition of the in45 cubation media. 46 Interestingly, a neuroprotective action against NMDA-induced 47 excitotoxicity, assessed by PI uptake, produced by both adenosine 48 A1 and cannabinoids CB1 receptors activation was only observed in 49 the CA3 area, which was the most susceptible to NMDA cytotoxic50 ity, but not the CA1 area. Since both adenosine A1 and cannabinoids 51 CB1 receptors are expressed at CA3 and CA1 areas – not only pre52 synaptically but also postsynaptically, at glutamatergic pyramidal 53 neurons, which also express glutamate NMDA receptors, and at 54 GABAergic interneurons (Mackie, 2005; Ochiishi et al., 1999) – the 55 absence of protective effect of A1 and CB1 receptors on NMDA56 induced cell injury at the CA1 area might reflect different receptor 57 expression/distribution between the two areas. 58 The robust additive inhibitory effect of combined A1 and CB1 re59 ceptor agonists on the NMDA-mediated excitotoxicity suggests that 60 a relevant neuroprotective role is played by A1 and CB1 receptors 61 against excitotoxic damage at the hippocampus. CB1 and A1 recep62 tors might use complementary mechanisms to afford neuroprotection 63 against NMDA-mediated neurotoxic insults since CB1 receptor64 mediated protection against NMDA-induced cell death depends on 65
adenylyl cyclase and ryanodine receptor inhibition (Zhuang et al., 2005), whereas A1 receptor-mediated protection did not depend on cAMP levels (Oku et al., 2004) but might involve direct inhibition of NMDA receptors (Sebastião et al., 2001).
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5. Conclusions The results obtained in the present work indicate an additive and therefore independent combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-mediated excitotoxicity. This cumulative dampening of excitotoxicity is relevant since a potential therapeutical role of combined A1 and CB1 agonists would be helpful in promoting neuronal survival and mitigating secondary brain damage in pathologies with associated excitotoxicity, such as ischemia, trauma or neurodegenerative disorders.
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Acknowledgements A. Serpa received a scholarship from FCT (SFRH/BD/65112/ 2009). The authors are grateful to Prof. A.M. Sebastião and Prof. J. A. Ribeiro (Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal) for helpful suggestions and discussion.
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Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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Rapid communication
Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus Q1 André Serpa a, Isa Pinto a, Liliana Bernardino a, José F. Cascalheira a,b,* a b
CICS-UBI – Health Sciences Research Center, University of Beira Interior, Covilhã, Portugal Department of Chemistry, University of Beira Interior, Covilhã, Portugal
A R T I C L E
I N F O
Article history: Received 19 April 2015 Received in revised form 3 June 2015 Accepted 4 June 2015 Available online Keywords: Adenosine A1 receptor Cannabinoid CB1 receptor Excitotoxicity Hippocampus
A B S T R A C T
Both adenosine A1 and cannabinoid CB1 receptors trigger similar transduction pathways and protect against neurotoxic insults at the hippocampus, but their combined neuroprotective potential has not been investigated. We set forth to assess the combined action of A1 and CB1 receptors against glutamate NMDA receptor-mediated excitotoxicity at the hippocampus. Cell damage was assessed by measuring propidium iodide (PI) uptake and lactate dehydrogenase (LDH) activity released from cultured hippocampal slices exposed to NMDA. Exposure to NMDA (50 μM) for 1 h increased LDH activity by 92% ± 16%. WIN55212-2 (30 μM), a cannabinoid CB1 receptor agonist, decreased NMDA-induced LDH activity by 53% ± 10% while N6-cyclopentyladenosine (CPA, 100 nM), an adenosine A1 receptor selective agonist, decreased it by 37% ± 11%. The combined inhibitory effect of WIN55212-2 and CPA (88% ± 12%) did not differ from the sum of the individual inhibitory effects of each agonist (90% ± 15%) but was different from the effects of CPA or WIN55212-2 alone. An additive inhibitory effect of co-application of WIN55212-2 (30 μM) and CPA (100 nM) on NMDA (50 μM)-induced PI uptake was also observed in CA3 but not in CA1 area of the hippocampal slice. The results suggest that both CB1 and A1 receptors produce additive cumulative neuroprotection against NMDA-induced excitotoxicity in the hippocampus. © 2015 Published by Elsevier Ltd.
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1. Introduction Sustained activation of glutamate NMDA receptors, following traumatic or ischemic brain injury, or caused by the pathological mechanisms of neurodegenerative diseases, generates neuronal death due to excitotoxicity, a secondary brain damage mechanism which involves excessive release of the neurotransmitter glutamate (Cross et al., 2010). The Gi/o-protein coupled cannabinoid CB1 and adenosine A1 receptors are both expressed at high levels in the hippocampus, were they inhibit glutamatergic synaptic transmission (Serpa Q2 et al., 2009) and protect against neurotoxic insults (Sebastião et al., 2001; Zhuang et al., 2005). Given the similarity between transducing pathways operated by adenosine A 1 and cannabinoid CB 1 receptors, as well as the identical effects produced by both receptors on nerve cells, clarification of the combined activity of these receptors is particularly relevant, since the harmful effects of neurotoxic insults might be cumulatively dampened. Previous studies showed that acute co-activation of adenosine A1 and cannabinoid
* Corresponding author. Departamento de Química, Universidade da Beira Interior, Rua Marquês D’Ávila e Bolama, 6200 Covilhã, Portugal. Tel.: +351 275329259; fax: +351 275319730. E-mail address: [email protected] (J.F. Cascalheira).
CB1 receptors generates additive effects when inhibiting excitatory synaptic transmission and cAMP formation in the rat hippocampus (Serpa et al., 2009, 2015). We now further tested how the combined action of A1 and CB1 receptor agonists modulates NMDA-mediated excitotoxic insult at the rat organotypic hippocampal slice.
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2. Materials and methods 2.1. Organotypic hippocampal slice cultures The interface culture method was used to prepare hippocampal slice cultures from 7-day-old Wistar rats (for details see Bernardino et al., 2005). Animals were handled according to European Community guidelines and Portuguese law concerning animal care. Slices (350 μm coronal sections) were prepared and sets of six were positioned on porous (0.4 μm) insert PTFE membranes (30 mm diameter; Millipore Corp., Bedford, USA) and transferred to a 6-well culture tray (Corning Costar, Corning, NY, USA). To each well was added 1 mL culture medium, composed of Opti-minimal essential medium (50%), heat-inactivated horse serum (25%), Hank’s balanced salt solution (25%) (all from Invitrogen, Paisley, UK), supplemented with D-glucose (25 mM) and also contained penicillin
http://dx.doi.org/10.1016/j.neuint.2015.06.005 0197-0186/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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(100 U/mL) and streptomycin (100 μg/mL) (Invitrogen). Culture trays were incubated at 37 °C under 5% CO2 and 95% atmospheric air and culture medium was changed twice a week for 2 weeks. Twentyfour hours before the experiment, medium was replaced with 1 mL serum free Neurobasal medium containing L-glutamine (1 mM) and 20 μL B27 supplement (Invitrogen). Previous work showed that 30 μM WIN55212-2 and 100 nM CPA produced a nearly maximal activation of CB1 and A1 receptors, respectively, as assessed by inhibition of cAMP formation in the rat hippocampal slice (Serpa et al., 2015), and therefore we used these concentrations to test the agonist neuroprotective effects.
and the PI uptake that would be expected in the presence of CPA, WIN55212-2 and NMDA if the effect of CPA and WIN55212-2 were additive was calculated as: A + B − C; where A, B and C are, respectively, LDH activity/PI uptake obtained in the presence of CPA + NMDA, in the presence of WIN55212-2 + NMDA, and in the absence of CPA and WIN55212-2 but in the presence of NMDA. Significance of differences between mean values obtained in two different conditions, or when comparing means with zero, was evaluated by Student’s t-test. When more than two different conditions were simultaneously compared one-way ANOVA was used, followed by LSD post-hoc test.
2.2. Evaluation of NMDA-induced excitotoxicity in slice cultures
3. Results
At the end of each experiment, aliquots of medium were collected to assess LDH activity. LDH activity was measured at room temperature using a commercial kit (LDH cytotoxicity assay kit, Cayman Chemical, Ann Arbor, USA). Quantification of LDH activity was not affected by endogenously released NADH/NADPH, since failure to add lactate to the reaction solution abolished LDH activity. Cell damage in slice cultures was also evaluated by measuring the cell uptake of propidium iodide (PI). PI (2 μM final concentration) was added to the incubation medium 3 hours before exposure to drugs, for determination of basal PI uptake, and PI addition was renewed after subsequent medium changes. Cellular uptake of PI was assessed by measuring emitted fluorescence (630 nm; absorbance 493 nm) using an inverted fluorescence microscope (Axio Observer Z1, Carl Zeiss, Jena, Germany) equipped with a rhodamine filter and digital camera (F-View 2; Olympus, Hamburg, Germany) with 100 ms exposure time. Fluorescence microscopy images of each slice were digitally recorded before drug exposure (basal PI uptake) and 24 h after exposure to the NMDA challenge (total PI uptake). To quantify cell damage, the slice areas of interest were delineated (>90% of the entire area) using Image J program (1.47 g, NIH, USA) and densitometric measurements of the PI uptake were performed. To assess the maximal level of cell death slices were exposed to an adverse environment (4 °C) for 48 h after performing the experiments. Maximal level of cell death was determined with both PI uptake, for each hippocampal area, and LDH release.
3.1. Neuroprotective action of adenosine A1 and cannabinoids CB1 receptors co-activation on NMDA-induced cytotoxicity in the whole hippocampal slice assessed by LDH release Exposure to NMDA alone (50 μM) for 1 h increased the released LDH activity from 7.0 ± 1.3 nmol/min/mL (control) to 12.4 ± 2.3 nmol/min/mL (19% ± 4% of maximal LDH release) corresponding to a 92% ± 16% increase (n = 4, see Fig. 1). Further application of the cannabinoid CB1 receptor selective agonist WIN55212-2 (30 μM), from 45 min before the NMDA insult, decreased the NMDAinduced release of LDH activity by 53% ± 10% (n = 4). In the same way, the adenosine A1 receptor selective agonist CPA (100 nM), when
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2.3. Drugs The drugs (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55212-2), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), N6-cyclopentyladenosine (CPA), N-(piperidin-1-yl)-5-(4-iodophenyl)1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and N-methyl-D-aspartate (NMDA) were purchased from Tocris (Bristol, UK). Propidium iodide was purchased from Sigma. Stock solutions of WIN55212-2 (20 mM), DPCPX (50 μM) and AM251 (5 mM) were prepared in dimethyl sulfoxide (DMSO) while CPA (2 mM) and NMDA (10 mM) stock solutions were prepared in water. 2.4. Data analysis The values are expressed as mean ± SEM from n experiments. The effect of drug(s) on the NMDA-induced LDH activity was calculated, for each experiment, as: 100 × (N − D)/(N − C); where N is the LDH activity obtained in the presence NMDA, D the LDH activity obtained in the presence of the drug(s) plus NMDA and C is the LDH activity obtained in the control assay performed in the same conditions but in the absence of the drug(s) and NMDA. PI uptake obtained in the presence of drug(s) was expressed as percentage of the PI uptake corresponding to control slices incubated in the same conditions but in the absence of drug(s). The released LDH activity
Fig. 1. Combined neuroprotective effect of WIN55212-2 and CPA on cell death induced by NMDA insult at the rat hippocampus evaluated by released lactate dehydrogenase (LDH). Two weeks old cultured organotypic hippocampal slices were exposed to WIN55212-2 (30 μM) or its vehicle (control). After 30 min CPA (100 nM final concentration) or its vehicle (control) were applied and 15 min later NMDA (50 μM final concentration) or its vehicle (control) were added and incubation continued for 1 h. Medium was replaced to remove drugs and released LDH activity was quantified 24 h later. In each experiment five parallel assays were performed, corresponding respectively to: incubation with NMDA, NMDA + WIN55212-2, NMDA + CPA, NMDA + WIN55212-2 + CPA and incubation in the absence of drugs (control). Bars represent released LDH activity obtained (from left to right) in the absence of drugs (control), in the presence of NMDA (b), NMDA + WIN55212-2 (c), NMDA + CPA (d) and NMDA + WIN55212-2 + CPA; the dashed bar represents the released LDH activity that would be obtained if the effects of CPA and WIN55212-2 were additive. Data are mean ± SEM from 4 independent experiment runs at least in triplicate. *, **, ***: P < 0.05, P < 0.02 and P < 0.00005, respectively, vs NMDA (b) alone (oneway ANOVA followed by LSD test). #, ##: P < 0.05 and P < 0.02, respectively, vs WIN55212-2 (c) or CPA (d) alone (one-way ANOVA followed by LSD test). NS, non statistically different (P > 0.90, Student’s t-test).
Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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applied 15 min prior to NMDA, decreased the NMDA-induced release of LDH activity by 37% ± 11% (n = 4). Interestingly, when WIN55212-2 (30 μM) and CPA (100 nM) were applied together, prior to NMDA (50 μM), the released LDH activity was reduced nearly to the levels obtained in the absence of NMDA (see Fig. 1). The combined application of WIN55212-2 (30 μM) and CPA (100 nM) produced an 88% ± 12% (n = 4) inhibition of the NMDA-induced LDH release from cultured hippocampal slices, which was higher than the inhibition produced by either WIN55212-2 (30 μM) or CPA (100 nM) alone (see Fig. 1) but not different from the sum of the individual inhibitory effect of each agonist (90% ± 15%; n = 4), indicating additivity of effects of both agonists. Neither WIN55212-2 (30 μM) nor CPA (100 nM) modified basal (control) release of LDH activity. The presence of the adenosine A1 receptor selective antagonist DPCPX (50 nM) reduced the inhibitory effect of 100 nM CPA to 1% ± 5% (n = 3, P < 0.05, Student’s t-test, compared with the effect in the absence of DPCPX), whereas the cannabinoid CB1 receptor selective antagonist AM251 (10 μM) decreased the effect of 30 μM WIN55212-2 to 11% ± 2% (n = 3, P < 0.05, Student’s t-test, compared with the effect in the absence of AM251), indicating that the inhibitory effects of CPA and WIN55212-2 are mediated by adenosine A1 and cannabinoid CB1 receptors, respectively. 3.2. Combined neuroprotective action of adenosine A1 and cannabinoids CB1 receptors on NMDA-induced cell injury in different areas of the hippocampal slice evaluated by fluorescence microscopy Fig. 2A depicts the effect of NMDA (50 μM) application for 1 h on the PI uptake in different areas of the rat hippocampal slice; the areas analyzed were those defined by Amaral and Witter (1989). As can be observed (Fig. 2A), NMDA-induced cell injury was highest in the CA3 area, where the PI uptake obtained was increased to 291% ± 19% (32% ± 3% of maximal PI uptake, n = 4) of the control PI uptake obtained in the absence of NMDA, while for the CA1 area the PI uptake was increased to 187% ± 22% of the control (20% ± 2% of maximal PI uptake, n = 4). No significant effect of NMDA (P > 0.05) on PI uptake was observed in CA4 and dentate gyrus areas (Fig. 2A). As can be observed in Fig 2B, for the CA3 area, WIN55212-2 (30 μM) and CPA (100 nM) when applied simultaneously produced a decrease of the NMDA (50 μM)-induced PI uptake, which was higher than the decrease produced by either WIN55212-2 (30 μM) or CPA (100 nM) alone but which was not different from the sum of the individual effects of each agonist. Note that this effect of WIN55212-2 and CPA was specific for the CA3 area. In CA1 area, the PI uptake obtained in the presence of NMDA (50 μM) alone (187% ± 22% of control PI uptake obtained in the absence of NMDA, n = 4) was not different from the PI uptake obtained in slices incubated with NMDA but in the presence of WIN55212-2 (30 μM), CPA (100 nM) or WIN55212-2 (30 μM) plus CPA (100 nM), respectively 182% ± 22%, 214% ± 20% and 180% ± 19% of the control PI uptake (P > 0.05, when compared with NMDA alone, one-Way ANOVA followed by LSD test). 4. Discussion The present study provides evidence, for the first time, of an independent, cumulative inhibitory effect of adenosine A 1 and cannabinoid CB1 receptors against NMDA-mediated excitotoxic insult at the rat hippocampus. Previous studies showed that, when applied alone, both A1 and CB1 receptor agonists decrease glutamatedependent neuronal activity and injury in the hippocampus (Sebastião et al., 2001; Zhuang et al., 2005). In the present work we observed that both A1 and CB1 agonists not only had individual neuroprotective action but also cumulatively dampened NMDAmediated excitotoxicity, with an additive combined effect higher than the one obtained activating each receptor alone. These results are
Fig. 2. Combined neuroprotective effect of WIN55212-2 and CPA on cell injury produced by NMDA insult at the rat hippocampus assessed by propidium iodide (PI) uptake. Two weeks old cultured organotypic hippocampal slices were exposed to WIN55212-2 (30 μM) or its vehicle (control). After 30 min CPA (100 nM final concentration) or its vehicle (control) were applied and 15 min later NMDA (50 μM final concentration) or its vehicle (control) were added and incubation continued for 1 h. Medium was replaced to remove drugs and incubation continued for a 24 h period. PI (2 μM final concentration) was added 3 hours before exposure to drugs and after medium replacement. In each experiment five parallel assays were performed, corresponding respectively to incubation with NMDA, NMDA + WIN55212-2, NMDA + CPA, NMDA + WIN55212-2 + CPA and incubation in the absence of drugs (control). Cellular uptake of PI was assessed by measuring emitted fluorescence before drugs addition (basal PI uptake) and 24 h after exposure to NMDA (total PI uptake). After subtracting basal PI uptake to total PI uptake, the net PI uptake was expressed as % of the net PI uptake obtained for control slices. (A) Bars represent PI uptake obtained in the absence (open bars) or in the presence of NMDA (solid bars) corresponding to (from left to right) the CA3, CA1, CA4 and dentate gyrus (DG) areas of the rat hippocampal slice. (B) Bars represent PI uptake obtained (from left to right) in the absence of drugs (control), in the presence of NMDA, NMDA + WIN55212-2, NMDA + CPA and NMDA + WIN55212-2 + CPA; the open bar represents the PI uptake that would be obtained if the effects of CPA and WIN55212-2 were additive. Data are mean ± SEM from 4 independent experiment runs at least in triplicate. §, §§, §§§: P < 0.05, P < 0.01 and P < 0.0001, respectively, vs NMDA (b) alone (oneway ANOVA followed by LSD test). #: P < 0.05 vs NMDA + WIN55212-2 (c) or NMDA + CPA (d) (one-way ANOVA followed by LSD test). NS, non statistically different (P > 0.9, Student’s t-test). *, **; P < 0.05 and P < 0.0001, respectively, vs control (Student’s t-test).
Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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1 in agreement with previous studies where acute co-activation of 2 adenosine A1 and cannabinoid CB1 receptor have been shown to generate additive effects in inhibiting glutamatergic synaptic trans3 mission and cAMP formation in the rat hippocampus (Serpa et al., 4 2009, 2015). 5 The combined additive neuroprotective action of adenosine A1 6 and cannabinoid CB1 receptors on NMDA-induced excitotoxicity was 7 evident when cell injury was assessed either by measuring LDH 8 release – which reflects cell necrosis and late apoptosis – or quan9 tifying PI uptake – which measures mainly cell necrosis and late 10 not only apoptosis but also early apoptosis in a smaller degree 11 (Parhamifar et al., 2013; Vitale et al., 1993). Although the com12 bined application of WIN55212-2 and CPA nearly prevented NMDA13 induced LDH release from hippocampal slices (88% decrease of LDH 14 release), the decrease in NMDA-induced PI uptake in CA3 area pro15 duced by both agonists was lower (47%). This difference might result 16 from the fact that LDH release reflects cell injury occurring in the 17 whole hippocampal slice, while PI uptake analysis is restricted to 18 superficial cell layers – less exposed to protective/trophic factors 19 released by neighboring cells than cells in the bulk of the slice – 20 that might have a lower ability to recover from NMDA-induced 21 damage upon activation of A1 and CB1 receptors. 22 Using PI fluorescence microscopy we were able to analyze NMDA23 induced cell injury in different hippocampal regions. The NMDA24 induced excitotoxic effect varied across different hippocampal areas, 25 the NMDA effect being stronger in CA3 area than in the CA1 area, 26 while no excitotoxic effect was observed at either CA4 or dentate 27 gyrus, indicating different susceptibilities of the neuronal popula28 tions across different hippocampal areas. The extent of NMDA29 induced cell injury in CA3 and CA1 areas observed in the present 30 work was similar to that reported in Sprague–Dawley rats in a pre31 vious study using the same NMDA concentration and exposure 32 period (Mayer et al., 2002), however they reported a significant neu33 rotoxic effect of NMDA in dentate gyrus, which was not observed 34 neither in the present work nor in a previous study also using Wistar 35 rats (Adamchik and Baskys, 2000). The higher extent of NMDA36 induced cell damage in CA3 than in CA1 area observed in the present 37 work and by Mayer et al. (2002), in contrasts with most previous 38 studies reporting CA1 area as the most susceptible to NMDA39 40 Q3 induced cytotoxicity – see Bruce et al. (1995), Ikegaya and Matsuki Q441Q5 Q6 (2002), Kristensen et al. (2001) and Vornov et al. (1991) (reviewed and cited respectively as references [9], [232], [11] and [12] 42 in Noraberg et al., 2005). The discrepancies between the results ob43 tained by the different studies may probably reflect differences in 44 the NMDA insult protocol, rat strains and/or composition of the in45 cubation media. 46 Interestingly, a neuroprotective action against NMDA-induced 47 excitotoxicity, assessed by PI uptake, produced by both adenosine 48 A1 and cannabinoids CB1 receptors activation was only observed in 49 the CA3 area, which was the most susceptible to NMDA cytotoxic50 ity, but not the CA1 area. Since both adenosine A1 and cannabinoids 51 CB1 receptors are expressed at CA3 and CA1 areas – not only pre52 synaptically but also postsynaptically, at glutamatergic pyramidal 53 neurons, which also express glutamate NMDA receptors, and at 54 GABAergic interneurons (Mackie, 2005; Ochiishi et al., 1999) – the 55 absence of protective effect of A1 and CB1 receptors on NMDA56 induced cell injury at the CA1 area might reflect different receptor 57 expression/distribution between the two areas. 58 The robust additive inhibitory effect of combined A1 and CB1 re59 ceptor agonists on the NMDA-mediated excitotoxicity suggests that 60 a relevant neuroprotective role is played by A1 and CB1 receptors 61 against excitotoxic damage at the hippocampus. CB1 and A1 recep62 tors might use complementary mechanisms to afford neuroprotection 63 against NMDA-mediated neurotoxic insults since CB1 receptor64 mediated protection against NMDA-induced cell death depends on 65
adenylyl cyclase and ryanodine receptor inhibition (Zhuang et al., 2005), whereas A1 receptor-mediated protection did not depend on cAMP levels (Oku et al., 2004) but might involve direct inhibition of NMDA receptors (Sebastião et al., 2001).
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5. Conclusions The results obtained in the present work indicate an additive and therefore independent combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-mediated excitotoxicity. This cumulative dampening of excitotoxicity is relevant since a potential therapeutical role of combined A1 and CB1 agonists would be helpful in promoting neuronal survival and mitigating secondary brain damage in pathologies with associated excitotoxicity, such as ischemia, trauma or neurodegenerative disorders.
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Acknowledgements A. Serpa received a scholarship from FCT (SFRH/BD/65112/ 2009). The authors are grateful to Prof. A.M. Sebastião and Prof. J. A. Ribeiro (Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal) for helpful suggestions and discussion.
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Please cite this article in press as: André Serpa, Isa Pinto, Liliana Bernardino, José F. Cascalheira, Combined neuroprotective action of adenosine A1 and cannabinoid CB1 receptors against NMDA-induced excitotoxicity in the hippocampus, Neurochemistry International (2015), doi: 10.1016/j.neuint.2015.06.005
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